Bottom Line:
Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines.A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control.These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.

Background: A malaria vaccine that targets the sporozoite/liver stage parasites could potentially prevent blood stage infection and the associated clinical symptoms. Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines. Cell-traversal protein for ookinetes and sporozoites (CelTOS) is a highly conserved antigen involved in sporozoite motility and hepatocyte invasion and has been shown to induce significant IFN-γ production in PBMCs from radiation-attenuated sporozoite-immunized malaria-naïve individuals. The aim of this study was to ascertain whether such CelTOS-specific recall responses are also induced in individuals with natural exposure to Plasmodium falciparum.

Methods: Ex vivo IFN-γ responses to 15mer overlapping peptide pools covering the entire sequence of CelTOS and five other candidate antigens, CSP, AMA1, MSP1, TRAP and LSA1, were characterized using PBMCs from 35 malaria exposed adults. Responses to four CelTOS peptide pools (CelTp1, CelTp2, CelTp3 and CelTp4), a pool containing peptides from the entire CelTOS antigen (CelTTp), and pools comprised of overlapping peptides from each of the other five malaria antigens were assessed by ex vivo ELISpot assay. A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control.

Results: Of the 35 volunteers tested, five had positive IFN-γ recall responses against the four different CelTOS pools while four volunteers made responses against the CelTTp pool; six volunteers were, therefore, positive with CelTOS. By contrast, six volunteers responded to AMA1, seven to LSA1, 15 to MSP1 and two volunteers responded against CSP and TRAP.

Conclusions: These results suggest natural malaria transmission induces CelTOS-specific ex vivo IFN-γ in Ghanaian adults and that the frequency of these responses was similar to those of other previously characterized malaria antigens. These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.

Fig2: Proportion of IFN-γ positive responders to the four separate CelTOS pools. The absolute number of responders for each pool has been expressed as a proportion of the total number of volunteers (35).

Mentions:
The magnitude of IFN-γ responses (sfc/m) to the CelTOS 15mer peptide pools CelTp1, CelTp2, CelTp3 and CelTp4 is shown in Figure 1, and the proportion of volunteers that responded to each pool is presented in Figure 2. Overall, five of the 35 (14%) study volunteers (v1, v4, v13, v16 and v30) were positive to at least one of the CelTOS peptide pools. Volunteer v28 had high activities in both test and control wells and was therefore not considered positive. Four of the five positive volunteers had positive activities to CelTp1 (v1, v4, v13 and v30), two of these were also positive with CelTp2 (v1 and v16) and one (v1) was positive with CelTp3. No activity was detected to CelTp4. The highest response was that of volunteer v1 to CelTp3 (64 sfc/m) and this volunteer also had the most positive responses (against CelTp1, CelTp2 and CelTp3, Figure 1). The remaining 30 volunteers did not respond to any of the CelTOS peptide pools, although they responded positively to Con A and CEF, except for v26 who responded only to CEF (Additional file 1). PBMCs from two malaria-naïve donors that were repeatedly stimulated with the same parasite peptides pools did not make any antigen-specific responses, though these cells also responded to stimulation with CEF controls (Additional file 2).Figure 1

Fig2: Proportion of IFN-γ positive responders to the four separate CelTOS pools. The absolute number of responders for each pool has been expressed as a proportion of the total number of volunteers (35).

Mentions:
The magnitude of IFN-γ responses (sfc/m) to the CelTOS 15mer peptide pools CelTp1, CelTp2, CelTp3 and CelTp4 is shown in Figure 1, and the proportion of volunteers that responded to each pool is presented in Figure 2. Overall, five of the 35 (14%) study volunteers (v1, v4, v13, v16 and v30) were positive to at least one of the CelTOS peptide pools. Volunteer v28 had high activities in both test and control wells and was therefore not considered positive. Four of the five positive volunteers had positive activities to CelTp1 (v1, v4, v13 and v30), two of these were also positive with CelTp2 (v1 and v16) and one (v1) was positive with CelTp3. No activity was detected to CelTp4. The highest response was that of volunteer v1 to CelTp3 (64 sfc/m) and this volunteer also had the most positive responses (against CelTp1, CelTp2 and CelTp3, Figure 1). The remaining 30 volunteers did not respond to any of the CelTOS peptide pools, although they responded positively to Con A and CEF, except for v26 who responded only to CEF (Additional file 1). PBMCs from two malaria-naïve donors that were repeatedly stimulated with the same parasite peptides pools did not make any antigen-specific responses, though these cells also responded to stimulation with CEF controls (Additional file 2).Figure 1

Bottom Line:
Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines.A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control.These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.

Background: A malaria vaccine that targets the sporozoite/liver stage parasites could potentially prevent blood stage infection and the associated clinical symptoms. Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines. Cell-traversal protein for ookinetes and sporozoites (CelTOS) is a highly conserved antigen involved in sporozoite motility and hepatocyte invasion and has been shown to induce significant IFN-γ production in PBMCs from radiation-attenuated sporozoite-immunized malaria-naïve individuals. The aim of this study was to ascertain whether such CelTOS-specific recall responses are also induced in individuals with natural exposure to Plasmodium falciparum.

Methods: Ex vivo IFN-γ responses to 15mer overlapping peptide pools covering the entire sequence of CelTOS and five other candidate antigens, CSP, AMA1, MSP1, TRAP and LSA1, were characterized using PBMCs from 35 malaria exposed adults. Responses to four CelTOS peptide pools (CelTp1, CelTp2, CelTp3 and CelTp4), a pool containing peptides from the entire CelTOS antigen (CelTTp), and pools comprised of overlapping peptides from each of the other five malaria antigens were assessed by ex vivo ELISpot assay. A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control.

Results: Of the 35 volunteers tested, five had positive IFN-γ recall responses against the four different CelTOS pools while four volunteers made responses against the CelTTp pool; six volunteers were, therefore, positive with CelTOS. By contrast, six volunteers responded to AMA1, seven to LSA1, 15 to MSP1 and two volunteers responded against CSP and TRAP.

Conclusions: These results suggest natural malaria transmission induces CelTOS-specific ex vivo IFN-γ in Ghanaian adults and that the frequency of these responses was similar to those of other previously characterized malaria antigens. These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.